When a blood vessel wall is damaged and subendothelial tissue is exposed to blood flow, platelets in the blood flow rapidly adhere to the subendothelial tissue. The adhesion requires a human von Willebrand factor (hereinafter simply referred to as “vWF”) in plasma. The vWF triggers a series of platelet activation steps, such as platelet aggregation and a release of intracellular granules, and then, formed thrombi lead hemostasis. In general, the vWF is secreted from a vascular endothelium to blood as a macromolecule having a molecular weight of more than 20,000 kDa, and cleaved by a metalloprotease, vWF-cleaving protease, into multimers having molecular weights of 500 to 20,000 kDa, which circulate through the blood. When a disease occurs (i.e., when a high shear stress is caused by occlusion or the like), the conformation of the vWF changes to an expanded structure. It is known that the expanded vWF has a high platelet aggregation activity, and the expanded vWF is liable to be degraded by the vWF-cleaving protease. It is considered that when the enzyme activity is lowered for some reason, “unusually large” vWF molecules are overproduced in the blood and efficiently bind to platelets and, as a result, the platelet aggregation in blood vessels is promoted to form thrombi in microcirculation. Such thrombus formation involved in platelets is essential for physiological hemostatic mechanisms. However, thrombi cause thrombotic diseases (such as cardiac infarction, cerebral infarction, or cerebral thrombosis), which are a major cause of death and a serious problem in an aging society.
It has been clarified that the vWF-cleaving protease is involved in thrombotic thrombocytopenic purpura (hereinafter simply referred to as “TTP”) which is extremely severe and has a high degree of fatality, that an autoantibody which inhibits the vWF-cleaving protease activity is produced in acute and sporadic TTP, and that the vWF-cleaving protease activity is inactive in familial TTP. Although a part of the vWF-cleaving protease was purified in 1996 (non-patent reference 1), the whole thereof was not identified until 2001. Because the vWF-cleaving protease exhibits its enzyme activity only in the presence of 1.5 mol/L urea/5 mmol/L Tris buffer (pH 8.0) in vitro, it was difficult to identify the vWF-cleaving protease as a substance. Recently, the plasma vWF-cleaving protease was purified (non-patent references 2 and 3). Further, cDNA thereof was cloned, and the gene, which belongs to an ADAMTS (a disintegrin like and metalloprotease with thrombospondin type 1 motif) family, was named ADAMTS13 (non-patent references 4 and 5). In the same period of time, it was clarified that the vWF-cleaving protease activity was significantly lowered in familiar TTP, due to a mutation of the vWF-cleaving protease gene ADAMTS13 (non-patent reference 6).
The vWF-cleaving protease activity was measured by detecting the large vWF multimers, using a combination of an SDS-agarose electrophoresis and autoradiography or Western blotting (non-patent reference 1). However, this measuring method contains complicated steps, and thus, is not a commonly used clinical laboratory test. For example, in this measuring method, a protease-free vWF is required, the procedure takes 3 days, and the measured values often vary in accordance with laboratories.
Recently, a method of measuring the vWF-cleaving protease activity, comprising the steps of expressing a partial region of an A2 domain (a site to be cleaved by the vWF-cleaving protease) of the vWF in Escherichia coli using genetic recombination techniques, mixing the recombinant protein with a sample derived from a patient for a predetermined period, to cleave the A2 domain by the vWF-cleaving protease contained in the sample, and detecting the cleaved products by a combination of SDS electrophoresis and Western blotting, was developed (non-patent reference 7). However, this method also contains complicated steps, such as a preparation of the recombinant protein or electrophoresis, and thus, it is difficult to use this method in most laboratories.
Idiopathic thrombocytopenic purpura (ITP) is a disease in which characteristic symptoms and clear causes of disease are unknown, and thrombocytopenia occurs by platelet destruction promoted by immunological mechanisms. In most cases, ITP is considered an autoimmune disease caused by an autoantibody against a platelet. As an antigen recognized by an anti-platelet antibody derived from a patient suffering from ITP, a platelet membrane protein GPIIb-IIIa was identified, and many methods for detecting an antibody specific for this protein were developed. Among these methods, an antigen capture assay using a monoclonal antibody against GPIIb-IIIa is widely used. It is known that this method shows a high specificity with a little false positive. However, most monoclonal antibodies which may be used in this method are not commercially available, and complicated steps, such as a collection of platelets or a dissolution of platelets, are required, and thus, the development of a convenient kit and standardization are required.